WO2002014858A2 - Method for producing and identifying appropriate effectors of target molecules using substance libraries - Google Patents

Method for producing and identifying appropriate effectors of target molecules using substance libraries Download PDF

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Publication number
WO2002014858A2
WO2002014858A2 PCT/EP2001/009103 EP0109103W WO0214858A2 WO 2002014858 A2 WO2002014858 A2 WO 2002014858A2 EP 0109103 W EP0109103 W EP 0109103W WO 0214858 A2 WO0214858 A2 WO 0214858A2
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Prior art keywords
library
substance
substances
binding
target
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PCT/EP2001/009103
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German (de)
French (fr)
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WO2002014858A3 (en
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Stefan Kienle
Norbert Windhab
Christoph BRÜCHER
Karsten Kuhn
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Nanogen Recognomics Gmbh
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Priority to US10/362,030 priority Critical patent/US20040038300A1/en
Priority to EP01976060A priority patent/EP1387853A2/en
Priority to JP2002519936A priority patent/JP2004510134A/en
Publication of WO2002014858A2 publication Critical patent/WO2002014858A2/en
Publication of WO2002014858A3 publication Critical patent/WO2002014858A3/en

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention relates to a method for the phenomenological description of target substances using characterized substance libraries and a method for selecting components of a combinatorial substance library for drug screening as well as the characterized and combinatorially generated substance libraries themselves.
  • the synthetically produced test substances are selected so that their physicochemical properties, such as. B. the lipophilicity parameter log P, the acid constant pK a or the solubility L, homogeneously cover the largest possible property space [H. Matter, J. Med. Chem. 1997, 40, 1219-1229].
  • a smaller group of potentially active substances is then selected from this library, which is as diverse as possible and therefore either incomplete or extremely extensive, by biological screening, often by simple binding assays.
  • defined compound libraries with restricted diversity are then synthesized and their biological activity against the target is examined. This results in one or more lead structures that are validated in further in vitro and in vivo assays.
  • ADMET absorbability
  • D distributability
  • M degradability
  • E excretibility
  • High throughput screening offers the handling and evaluation of a large number of substances to be tested.
  • active substances can be identified either by direct analysis of the immobilized substance or by coding the solid phase ⁇ beads during the synthesis and reading out the code of active beads.
  • the biological high-throughput screening of combinatorial compound libraries in solution requires a multi-stage deconvolution and a complex synthesis of increasingly defined sub-libraries.
  • the object of the present invention is to provide a targeted method for the development of biologically and / or chemically active substances without having to resort to a rational molecular design.
  • potentially binding-active substances are understood to be molecules which can interact with other compounds, in particular with nucleic acids, proteins or peptides.
  • nucleic acids, proteins or peptides include e.g. B. low molecular weight substances such as carboxylic acids, amines, esters, aldehydes, ketones, acetals and heterocycles such as alkaloids, and lipids, saccharides, steroids and others Natural products, but it is also possible to use peptides and proteins, such as antibodies or peptoids and their homo- or heterodimers or multimers, or known agonists and antagonists of proteins.
  • the library is supplemented by a selection of potentially binding substances that have predetermined physicochemical properties, such as. B. size, lipophilicity or polarity as homogeneously as possible, cover a wide range of properties.
  • a suitable selection can e.g. B. based on (J. M. Blaney, E. J. Martin, Current Opinion in Chemical Biology 1997, 1, 54-59; H. Matter, J. Med. Chem. 1997, 40, 1219-1229).
  • test substances are characterized with test substances and thus characterized. All known proteins, polypeptides or nucleic acids, of which the structure of at least one agonist or one antagonist is known, can be used as the test substance.
  • Preferred test substances are already well-characterized nucleic acids, proteins and peptides, such as. B. receptors, antibodies, enzymes, transcription factors, ion channels or coding or gene regulatory DNA sequences, such as promoters or operators.
  • Particularly preferred test substances are all compounds which are known to the person skilled in the art as therapeutic targets.
  • the library is characterized by determining the binding pattern of the test substances with the binding-active substances of the library.
  • the contact can be made either homogeneously in solution or heterogeneously with test substance immobilized on the solid phase.
  • the test substance and the library to be characterized are each dissolved in a suitable solvent and brought into interaction. It is advantageous under defined conditions, such as. B. a precisely defined concentration of the test substance and the potentially binding substances or z. B. the reproducible use of physiological solution conditions to work. Then samples are taken from the solution removed and the static binding pattern of the test substance on the decrease in the concentration of the binding-active substances from the library z. B. using electrospray ionization mass spectrometry (ESI-MS), nanospray ESI-MS, matrix assisted laser desorption ionization - mass spectrometry (MALDI) or time of flight secondary ion mass spectrometry (TOF-SIMS). Before the binding pattern is determined by mass spectrometry, the sample taken can be separated chromatographically, but the sample can also be examined directly by mass spectrometry.
  • ESI-MS electrospray ionization mass spectrometry
  • MALDI matrix assisted laser desorption ionization - mass
  • the contacting preferably takes place in homogeneous solution in a dialysis unit.
  • all potentially binding substances of the library to be characterized are present in a defined initial concentration, which are preferably far above the concentration of the test substance.
  • samples can be taken at defined time intervals and the intensities of the mass signals of the individual components of the library can be determined in a time-resolved manner using ESI-MS, Nanospray-ESI-MS, MALDI or TOF-SIMS.
  • the samples can be examined either without further purification or after preferably chromatographic purification.
  • a binding-active component of the library to be characterized and the test substance can be separated either in the spectrometer or preferably online chromatographically. If the concentration of the library falls below a certain value as dialysis progresses, a non-competitive situation arises in which less active substances can also bind.
  • This temporally dynamic test sequence provides additional information on the basis of the concentration gradient generated with regard to the potentially binding substances in the course of the measurement over time.
  • test substances are applied to a solid phase such as e.g. B. bound magnetic polymer beads.
  • the library to be characterized is added to the immobilized test substance in defined, different concentrations, so that non-competitive as well as competitive systems are created. After the solid phase has been separated off with the bound active components of the library, either the components of the MSL remaining in the solution or the bound components after elution from the solid phase are determined using mass spectrometric methods.
  • the library can be characterized by repeatedly determining the static or the temporally-dynamic binding pattern of the binding-active substances from the library with different test substances. H. can be characterized in the sense of this invention. The more test substances used to train the library, the better the binding behavior of the library is characterized.
  • test substance can be problem-oriented, so preferred test substances are well-characterized native or artificial proteins if protein library target substances are to be examined with this library.
  • Pattern recognition is used as a mathematical tool for analyzing this data (K. Backhaus, B. Erichson, W. Plinke, R. Wieber, Multivariate Analysis Methods, 9th edition, 2000, Springer Verlag; DT Stanton, TW Morris , S. Roychoudhury, CN Parker, J. Chem. Inf. Comput. Sci. 1999, 39, 21-27; PC Jurs, Science 1986, 232, 1219-1224). If a characterized library of n potentially binding-active components is characterized with m test substances, then a defined point in an n-dimensional space that is spanned by the n components can be assigned to each test substance. The selected test substances ideally cover the entire spanned n-dimensional space that represents the components of the characterized library.
  • a target substance is exposed to a characterized library according to the invention, its binding pattern can be determined analogously to the methods described for the test substances.
  • the binding pattern determined describes the target substance phenomenologically. Are z.
  • the target substance can be assigned a specific point in n-dimensional space that is spanned by the individual components.
  • Chemical target substances or test substances with a similar binding behavior are assigned to closely spaced points in the library-specific n-dimensional space.
  • the invention further relates to a method for the phenomenological description of target substances using a characterized library of potentially binding active substances according to the invention, in which the target compound to be investigated is brought into interaction with the characterized library and subsequently the binding pattern of the target substance generated by the specific interactions is determined becomes. Based on the determined binding pattern, a point in the n-dimensional space of the characterized library is assigned to the target substance according to the method described above, which point reflects the binding behavior of the target substance. Now the test substances can be determined, which are represented by neighboring points in the n-dimensional space of the characterized library and therefore show a similar binding behavior.
  • the known agonists or antagonists of the test substances can then be used to identify physicochemical descriptors which are conducive to specific binding with the target substance under investigation.
  • a great advantage of the present invention is that a newly identified target structure, for which neither agonists, antagonists or other binding partners need to be known, can be correlated with a group of test substances which have structurally similar features and / or physico-chemically similar properties. Conversely, this means that, based on the structural characteristics and physicochemical properties of the known agonists, antagonists and non-binders of the similar test substances, conclusions can be drawn about the physicochemical and structural requirements of potential agonists and antagonists of the investigated target structure.
  • the method according to the invention consequently enables the targeted selection of physicochemical descriptors that can be used in the search for active substances (effectors) to an unknown target substance, without having to rely on a rational molecular design must be used.
  • gene-regulatory active DNA sequences such as. z. B. promoters or operators.
  • the descriptors identified by the method described above can e.g. B ; , to produce a combinatorial-synthetically generated bank (JM Blaney, EJ Martin, Current Opinion in Chemical Biology 1997, 1, 54-59) from potential effectors.
  • B Structural elements that promote the membrane passage of active ingredients, or structural elements that improve the biodegradability or excretibility (PJ Sinko, Current Opinion in Drug Discovery & Development 1999, 242-48).
  • effector in the sense of this invention is a biologically or chemically active substance which specifically interacts with the target substance to be investigated and influences its function. Effectors are e.g. B. inhibitors, activators or inducers of enzymes, coenzymes, transcription factors or repressors.
  • Another object of the invention is thus the use of descriptors, which were determined using a characterized library according to the invention, for the production of a library from potential effectors, and the target-generated libraries for the identification of suitable effectors of the target substance to be examined.

Abstract

The invention relates to a method for the phenomenological description of target substances using described substance libraries. The invention also relates to a method for selecting the components of a combinatorial substance library for the purpose of active ingredient screening and to the described and combinatorially produced substance libraries as such.

Description

Beschreibungdescription
Verfahren zur Herstellung und Ermittlung geeigneter Effektoren von Zielmolekülen mit SubstanzbibliothekenProcess for the production and determination of suitable effectors of target molecules with substance libraries
Die vorliegende Erfindung betrifft ein Verfahren zur phänomenologischen Beschreibung von Zielsubstanzen unter Verwendung charakterisierter Substanzbibliotheken und ein Verfahren zur Auswahl von Komponenten einer kombinatorischen Substanzbibliothek für das Wirkstoffscreening sowie die charakterisierten und kombinatorisch erzeugten Substanzbibliotheken selbst.The present invention relates to a method for the phenomenological description of target substances using characterized substance libraries and a method for selecting components of a combinatorial substance library for drug screening as well as the characterized and combinatorially generated substance libraries themselves.
Die Suche nach therapeutisch oder diagnostisch aktiven Wirkstoffen beginnt im allgemeinen mit der Identifizierung und Validierung geeigneter Target-Substanzen. Die Untersuchung solcher biologischen Targets im Hinblick auf potentiell pharmakologisch wirksame Substanzen gestaltet sich häufig als sehr schwierig.The search for therapeutically or diagnostically active substances generally begins with the identification and validation of suitable target substances. The investigation of such biological targets with regard to potentially pharmacologically active substances is often very difficult.
Auf die Identifizierung eines geeigneten Targets folgt häufig, gemäß bekannter Methoden aus dem Stand der Technik, die gezielte, gegebenenfalls kombinatorische, Synthese von Einzelverbindungen oder Verbindungsbibliotheken [L. Van Hijfte, G. Marciniak, N. Froloff, J. Chromatogr. B 1999, 725, 3-15], die in in vitro oder in vivo Assays auf ihre biologische Aktivität gegen das Target untersucht werden.The identification of a suitable target is often followed, according to known methods from the prior art, by the targeted, optionally combinatorial, synthesis of individual compounds or compound libraries [L. Van Hijfte, G. Marciniak, N. Froloff, J. Chromatogr. B 1999, 725, 3-15], which are examined in in vitro or in vivo assays for their biological activity against the target.
Die synthetisch erzeugten Testsubstanzen werden so ausgewählt, daß ihre physikochemischen Eigenschaften, wie z. B. der Lipophilizitätsparameter log P, die Säurekonstante pKa oder die Löslichkeit L, einen möglichst großen Eigenschaftsraum homogen abdecken [H. Matter, J. Med. Chem. 1997, 40, 1219- 1229]. Aus dieser möglichst diversen, und damit entweder lückenhaften oder äußerst umfangreichen, Bibliothek von Verbindungen wird dann eine kleinere Gruppe von potentiell aktiven Substanzen durch biologisches Screening, oft durch einfache Bindungsassays, ausgewählt. Ausgehend von den spezifischen physikochemischen Eigenschaften dieser potentiell aktiven Substanzen werden dann wiederum definierte Verbindungsbibliotheken mit eingeschränkter Diversität synthetisiert und auf ihre biologische Aktivität gegen das Target untersucht. Daraus resultieren eine oder mehrere Leitstrukturen, die in weiteren in vitro und in vivo Assays validiert werden.The synthetically produced test substances are selected so that their physicochemical properties, such as. B. the lipophilicity parameter log P, the acid constant pK a or the solubility L, homogeneously cover the largest possible property space [H. Matter, J. Med. Chem. 1997, 40, 1219-1229]. A smaller group of potentially active substances is then selected from this library, which is as diverse as possible and therefore either incomplete or extremely extensive, by biological screening, often by simple binding assays. On the basis of the specific physicochemical properties of these potentially active substances, defined compound libraries with restricted diversity are then synthesized and their biological activity against the target is examined. This results in one or more lead structures that are validated in further in vitro and in vivo assays.
Zum Schluß werden die aktiven Substanzen auf ihre ADMET-Eigenschaften (ADMET steht für Absorbierbarkeit (A), Verteilbarkeit (D), Abbaubarkeit (M) , Ausscheidbarkeit (E) und Toxizität für Organismen oder Zellverbände) untersucht werden bevor die Substanzen in die klinische Untersuchungsphase übergehen.Finally, the active substances are examined for their ADMET properties (ADMET stands for absorbability (A), distributability (D), degradability (M), excretibility (E) and toxicity for organisms or cell groups) before the substances enter the clinical investigation phase pass.
Ein solches, auf dem „Try and Error'-Prinzip basierendes Verfahren ist entsprechend aufwendig und kostenintensiv in seiner Durchführung. Insbesondere sind eine große Anzahl von Einzelversuchen durchzuführen, um geeignete Bindungspartner des Targets zu identifizieren. Erst zu einem sehr späten Zeitpunkt der Entwicklungsphase kann eine weitere Optimierung der biologisch aktiven Substanzen erfolgen, deren Erfolg häufig für die Einsetzbarkeit, z. B. als pharmakologischer Wirkstoff, entscheidend ist.Such a method, which is based on the “try and error” principle, is correspondingly complex and costly to carry out. In particular, a large number of individual tests have to be carried out in order to identify suitable binding partners of the target. Only at a very late point in the development phase can further optimization of the biologically active substances take place, the success of which is often due to their applicability, e.g. B. as a pharmacological agent is crucial.
Die Handhabung und Bewertung einer größeren Anzahl von zu testenden Substanzen bietet das Hochdurchsatzscreening. Beim biologischen Hochdurchsatzscreening von kombinatorischen Verbindungsbibliotheken an fester Phase, die z. B. nach dem split-couple-recombine Verfahren hergestellt wurden, können aktive Substanzen entweder durch direkte Analyse der immobilisierten Substanz oder durch Codierung der Festphaseήkügelchen während der Synthese und Auslesen des Codes aktiver Kügelchen identifiziert werden. Das biologische Hochdurchsatzscreening von kombinatorischen Verbindungsbibliotheken in Lösung erfordert allerdings eine mehrstufige Dekonvolution und eine aufwendige Synthese immer definierterer Subbibliotheken. Häufig werden auch Einzelverbindungen hochparallel und automatisiert hergestellt und als Einzelverbindungen dem Hochdurchsatzscreening zugeführt, um aktive Substanzen direkt identifizieren zu können [D. L. Venton, C. P. Woodbury, Chemom. Intell. Lab. Syst. 1999, 48, 131- 150]. Die Unterscheidung zwischen potentiell aktiven Komponenten der Testverbindungen, die weiter bearbeitet werden, und nicht aktiven Komponenten muß dabei häufig willkürlich getroffen werden [G. W. Caldwell, Curr. Opin. Drug Discovery Dev. 2000, 3, 30-41]. Oft werden die biologisch aktivsten 30% der Komponenten einer Verbindungsbibliothek oder subjektiv wirkstoffähnliche Komponenten, bezüglich molekularem Grundgerüst und den funktioneilen Seitenketten, ausgewählt [B. Ladd, Mod. Drug Discovery 2000, 1 , 46-52].High throughput screening offers the handling and evaluation of a large number of substances to be tested. In biological high-throughput screening of combinatorial compound libraries on a solid phase, e.g. B. were produced by the split-pair recombine method, active substances can be identified either by direct analysis of the immobilized substance or by coding the solid phaseήbeads during the synthesis and reading out the code of active beads. The biological high-throughput screening of combinatorial compound libraries in solution, however, requires a multi-stage deconvolution and a complex synthesis of increasingly defined sub-libraries. Individual connections are also often produced in a highly parallel and automated manner and fed to high-throughput screening as individual connections in order to be able to identify active substances directly [DL Venton, CP Woodbury, Chemom. Intell. Lab. Syst. 1999, 48, 131-150]. The distinction between potentially active components of the test compounds, which are further processed, and inactive components must often be made arbitrarily [GW Caldwell, Curr. Opin. Drug Discovery Dev. 2000, 3, 30-41]. Often the most biologically active 30% of the components of a compound library or subjectively drug-like components are selected with regard to the molecular backbone and the functional side chains [B. Ladd, Mod. Drug Discovery 2000, 1, 46-52].
Aus diesen Gründen wird versucht neben den „Try and Error"- Methoden Verfahren zum rationalen Wirkstoffdesign zu entwickeln. Zu nenn sind hier vor allem das Molecular Modelling und die SAR-Analyse [A. Ajay, W. P. Walters, M. A. Murcko, J. Med. Chem. 1998, 41 , 3314-3324; E. Hodgkin, K. Andrews-Cramer, Mod. Drug Discovery 2000, 3, 55-60]. Eine Vielzahl von Computerprogrammen sind heutzutage dafür erhältlich [W. A. Warr, J. Chem. Inf. Comput. Sei. 1997, 37,134-140]. Mit Hilfe der rein rationalen Methoden zum zielgerichteten Design biologisch aktiver Substanzen konnten bisher allerdings nur wenige Wirkstoffe entwickelt werden, sie haben sich in der Praxis bisher nicht bewährt. Weiterhin muß für ein rationales Wirkstoffdesign das Target sehr gut charakterisiert, häufig muß sogar die dreidimensionale Struktur des Targets bekannt sein.For these reasons, attempts are being made to develop methods for rational drug design in addition to the “try and error” methods. Molecular modeling and SAR analysis are particularly worth mentioning [A. Ajay, WP Walters, MA Murcko, J. Med. Chem. 1998, 41, 3314-3324; E. Hodgkin, K. Andrews-Cramer, Mod. Drug Discovery 2000, 3, 55-60]. A variety of computer programs are available today [WA Warr, J. Chem. Inf 1997, 37, 134-140]. With the help of purely rational methods for the targeted design of biologically active substances, however, only a few active substances have so far been able to be developed, and they have so far not proven themselves in practice Target very well characterized, often even the three-dimensional structure of the target must be known.
Der vorliegenden Erfindung liegt die Aufgabe zu Grunde ein zielgerichtetes Verfahren zur Entwicklung von biologisch und/oder chemisch aktiven Substanzen zur Verfügung zu stellen, ohne auf ein rationales Moleküldesign zurückgreifen zu müssen.The object of the present invention is to provide a targeted method for the development of biologically and / or chemically active substances without having to resort to a rational molecular design.
Dazu wird eine erfindungsgemäße charakterisierte Bibliothek enthaltend eine große Anzahl von potentiell bindungsaktiven Substanzen verwendet. Als potentiell bindungsaktive Substanzen im Sinne dieser Erfindung werden Moleküle verstanden die mit anderen Verbindungen, insbesondere mit Nukleinsäuren, Proteinen oder Peptiden, in Wechselwirkung treten können. Dazu zählen z. B. niedermolekulare Stoffe, wie Carbonsäuren, Amine, Ester, Aldehyde, Ketone, Acetale und Heterocyclen, wie Alkaloide, und Lipide, Saccharide, Steroide sowie andere Naturstoffe, es können aber auch Peptide und Proteine, wie Antikörper oder Peptoide sowie deren Homo- oder Heterodimere bzw. -multimere oder bekannte Agonisten und Antagonisten von Proteinen verwendet werden.For this purpose, a characterized library according to the invention containing a large number of potentially binding substances is used. For the purposes of this invention, potentially binding-active substances are understood to be molecules which can interact with other compounds, in particular with nucleic acids, proteins or peptides. These include e.g. B. low molecular weight substances such as carboxylic acids, amines, esters, aldehydes, ketones, acetals and heterocycles such as alkaloids, and lipids, saccharides, steroids and others Natural products, but it is also possible to use peptides and proteins, such as antibodies or peptoids and their homo- or heterodimers or multimers, or known agonists and antagonists of proteins.
Zusätzlich wird die Bibiothek durch eine Auswahl von potentiell bindungsaktiven Substanzen ergänzt, die vorgegebene physikochemische Eigenschaften, wie z. B. Größe, Lipophilizität oder Polarität möglichst homogen, über einen weiten Eigenschaftsbereich, abdecken. Eine geeignete Auswahl kann z. B. anhand (J. M. Blaney, E. J. Martin, Current Opinion in Chemical Biology 1997, 1 , 54-59; H. Matter, J. Med. Chem. 1997, 40, 1219-1229) erfolgen.In addition, the library is supplemented by a selection of potentially binding substances that have predetermined physicochemical properties, such as. B. size, lipophilicity or polarity as homogeneously as possible, cover a wide range of properties. A suitable selection can e.g. B. based on (J. M. Blaney, E. J. Martin, Current Opinion in Chemical Biology 1997, 1, 54-59; H. Matter, J. Med. Chem. 1997, 40, 1219-1229).
Die Bibliothek aus poteniell bindungsaktiven Substanzen wird mit Testsubstanzen charakterisiert und damit charakterisiert. Als Testsubstanz kommen alle bekannten Proteine, Polypeptide oder Nukleinsäuren in Frage, von denen die Struktur mindestens eines Agonisten oder eines Antagonisten bekannt ist. Bevorzugte Testsubstanzen sind bereits gut charakterisierte Nukleinsäuren, Proteine und Peptide, wie z. B. Rezeptoren, Antikörper, Enzyme, Transkriptionsfaktoren, lonenkanäle oder auch codierende oder genregulatorische DNA-Sequenzen, wie Promotoren oder Operatoren. Besonders bevorzugte Testsubstanzen sind alle Verbindungen, die dem Fachmann als therapeutische Targets bekannt sind.The library of potentially binding substances is characterized with test substances and thus characterized. All known proteins, polypeptides or nucleic acids, of which the structure of at least one agonist or one antagonist is known, can be used as the test substance. Preferred test substances are already well-characterized nucleic acids, proteins and peptides, such as. B. receptors, antibodies, enzymes, transcription factors, ion channels or coding or gene regulatory DNA sequences, such as promoters or operators. Particularly preferred test substances are all compounds which are known to the person skilled in the art as therapeutic targets.
Die Charakterisierung der Bibliothek erfolgt über die Bestimmung der Bindungsmuster der Testsubstanzen mit den bindungsaktiven Substanzen der Bibliothek. Die Kontaktierung kann entweder in homogen in Lösung oder heterogen mit an fester Phase immobilisierter Testsubstanz erfolgen.The library is characterized by determining the binding pattern of the test substances with the binding-active substances of the library. The contact can be made either homogeneously in solution or heterogeneously with test substance immobilized on the solid phase.
Beim homogenen Assay werden die Testsubstanz und die zu charakterisierende Bibliothek, jeweils in einem geeigneten, Lösungsmittel gelöst und in Wechselwirkung gebracht. Vorteilhaft ist es unter definierten Bedingungen, wie z. B. einer genau definierten Konzentration der Testsubstanz und der potentiell bindungsaktiven Substanzen oder z. B. der reproduzierbaren Verwendung physiologischer Lösungsbedingungen, zu arbeiten. Anschließend werden Proben aus der Lösung entnommen und das statische Bindungsmuster der Testsubstanz über die Abnahme der Konzentration der bindungsaktiven Substanzen aus der Bibliothek z. B. mittels Elektrospray-Ionisierungs Massenspektrometrie (ESI-MS), Nanospray-ESI-MS, Matrix Assisted Laser Desorption lonization - Massenspektrometrie (MALDI) oder Time of Flight Sekundärionen-Massenspektrometrie (TOF-SIMS) bestimmt. Vor einer massensprktrometrischen Bestimmung des Bindungsmuster ist eine chromatographische Trennung der entnommenen Probe möglich, die Probe kann allerdings auch direkt massenspektrometrisch untersucht werden.In the homogeneous assay, the test substance and the library to be characterized are each dissolved in a suitable solvent and brought into interaction. It is advantageous under defined conditions, such as. B. a precisely defined concentration of the test substance and the potentially binding substances or z. B. the reproducible use of physiological solution conditions to work. Then samples are taken from the solution removed and the static binding pattern of the test substance on the decrease in the concentration of the binding-active substances from the library z. B. using electrospray ionization mass spectrometry (ESI-MS), nanospray ESI-MS, matrix assisted laser desorption ionization - mass spectrometry (MALDI) or time of flight secondary ion mass spectrometry (TOF-SIMS). Before the binding pattern is determined by mass spectrometry, the sample taken can be separated chromatographically, but the sample can also be examined directly by mass spectrometry.
Vorzugsweise findet die Kontaktierung in homogener Lösung in einer Dialyseeinheit statt. Zu Beginn liegen alle potentiell bindungsaktiven Substanzen der zu charakterisierenden Bibliothek in definierter Ausgangskonzentration, die vorzugsweise weit über der Konzentration der Testsubstanz liegen, vor. Somit binden vorrangig nur die bindungsaktivsten Substanzen der Bibliothek während die weniger aktiven Komponenten in dieser kompetitiven Situation keine Bindung eingehen. Im Laufe der Dialyse können nach definierten Zeitintervallen Proben entnommen und die Intensitäten der Massensignale der einzelnen Komponenten der Bibliothek mit ESI-MS, Nanospray-ESI-MS, MALDI oder TOF-SIMS zeitaufgelöst bestimmt werden. Die Proben können entweder ohne weitere Aufreinigung oder nach vorzugsweise chromatographischer Aufreinigung untersucht werden.The contacting preferably takes place in homogeneous solution in a dialysis unit. At the beginning, all potentially binding substances of the library to be characterized are present in a defined initial concentration, which are preferably far above the concentration of the test substance. As a result, only the most active substances in the library bind, while the less active components do not bind in this competitive situation. During the dialysis, samples can be taken at defined time intervals and the intensities of the mass signals of the individual components of the library can be determined in a time-resolved manner using ESI-MS, Nanospray-ESI-MS, MALDI or TOF-SIMS. The samples can be examined either without further purification or after preferably chromatographic purification.
Das Trennen einer bindungsaktiven Komponente der zu charakterisierenden Bibliothek und der Testsubstanz kann entweder im Spektrometer oder vorzugsweise online chromatographisch erfolgen. Unterschreitet die Konzentration der Bibliothek bei fortschreitender Dialyse einen bestimmten Wert, entsteht eine nicht-kompetitive Situation, bei der auch weniger aktive Substanzen binden können. Dieser zeitlichdynamische Versuchsablauf liefert zusätzliche Informationen aufgrund des erzeugten Konzentrationsgradienten bezüglich der potentiell bindungsaktiven Substanzen im zeitlichen Verlauf der Messung. Beim nicht-homogenen Assay werden Testsubstanzen an eine feste Phase wie z. B. magnetische Polymerkügelchen gebunden. Die zu charakterisierende Bibliothek wird in definierten, unterschiedlichen Konzentrationen zu der immobilisierten Testsubstanz gegeben, so daß nicht-kompetitive als auch kompetitive Systeme entstehen. Nach Abtrennen der festen Phase mit den gebundenen aktiven Komponenten der Bibliothek werden entweder die in der Lösung verbliebenen Komponenten der MSL oder die gebundenen Komponenten nach Elution von der festen Phase mit massenspektrometrischen Methoden bestimmt.A binding-active component of the library to be characterized and the test substance can be separated either in the spectrometer or preferably online chromatographically. If the concentration of the library falls below a certain value as dialysis progresses, a non-competitive situation arises in which less active substances can also bind. This temporally dynamic test sequence provides additional information on the basis of the concentration gradient generated with regard to the potentially binding substances in the course of the measurement over time. In the non-homogeneous assay, test substances are applied to a solid phase such as e.g. B. bound magnetic polymer beads. The library to be characterized is added to the immobilized test substance in defined, different concentrations, so that non-competitive as well as competitive systems are created. After the solid phase has been separated off with the bound active components of the library, either the components of the MSL remaining in the solution or the bound components after elution from the solid phase are determined using mass spectrometric methods.
Durch das wiederholte bestimmen des statischen oder des zeitlich-dynamisch Bindungsmusters der bindungsaktiven Substanzen aus der Bibliothek mit unterschiedlichen Testsubstanzen kann die Bibliothek charakterisiert werden, d. h. im Sinne dieser Erfindung charakterisiert werden. Je mehr Testsubstanzen zum Training der Bibliothek herangezogen werden desto besser ist das Bindungsverhalten der Bibliothek charakterisiert.The library can be characterized by repeatedly determining the static or the temporally-dynamic binding pattern of the binding-active substances from the library with different test substances. H. can be characterized in the sense of this invention. The more test substances used to train the library, the better the binding behavior of the library is characterized.
Die Auswahl der Testsubstanz kann problemorientiert erfolgen, so sind bevorzugte Testsubstanzen gut charakterisierte native oder artifizielle Proteine wenn mit dieser Bibliothek proteinische Zielsubstanzen untersucht werden sollen.The selection of the test substance can be problem-oriented, so preferred test substances are well-characterized native or artificial proteins if protein library target substances are to be examined with this library.
Als mathematisches Werkzeug zur Analyse dieser Daten wird die Mustererkennung, speziell die Clusteranalyse, eingesetzt (K. Backhaus, B. Erichson, W. Plinke, R. Wieber, Multivariate Analysemethoden, 9. Auflage, 2000, Springer Verlag; D. T. Stanton, T. W. Morris, S. Roychoudhury, C. N. Parker, J. Chem. Inf. Comput. Sei. 1999, 39, 21-27; P. C. Jurs, Science 1986, 232, 1219-1224). Wird eine charakterisierte Bibliothek aus n potentiell bindungsaktiven Komponenten mit m Testsubstanzen charakterisiert, dann kann jeder Testsubstanz ein definierter Punkt in einem n-dimensionalen Raum, der von den n Komponenten aufgespannt wird, zugeordnet werden. Die ausgewählten Testsubstanzen decken im Idealfall den gesamten aufgespannten n-dimensionale Raum der die Komponenten der charakterisierten Bibliothek repräsentiert punktuell ab.Pattern recognition, especially cluster analysis, is used as a mathematical tool for analyzing this data (K. Backhaus, B. Erichson, W. Plinke, R. Wieber, Multivariate Analysis Methods, 9th edition, 2000, Springer Verlag; DT Stanton, TW Morris , S. Roychoudhury, CN Parker, J. Chem. Inf. Comput. Sci. 1999, 39, 21-27; PC Jurs, Science 1986, 232, 1219-1224). If a characterized library of n potentially binding-active components is characterized with m test substances, then a defined point in an n-dimensional space that is spanned by the n components can be assigned to each test substance. The selected test substances ideally cover the entire spanned n-dimensional space that represents the components of the characterized library.
Wird eine Zielsubstanz einer erfindungsgemäßen charakterisierten Bibliothek ausgesetzt kann deren Bindungsmuster analog zu den für die Testsubstanzen beschriebenen Verfahren bestimmt werden. Das ermittelte Bindungsmuster beschreibt die Zielsubstanz dabei phänomenologiseh. Sind z. B. lipophile Regionen in der Zielsubstanz vorhanden binden entsprechende bindungsaktive Substanzen aus der charakterisierten Bibliothek, sind polare Regionen zugänglich werden polare Substanzen aus der Bibliothek gebunden. Durch das ermittelte Bindungsmuster kann der Zielsubstanz ein spezifischer Punkt im n-dimensionalen Raum, der durch die einzelnen Komponenten aufgespannt wird, zugeordnet werden.If a target substance is exposed to a characterized library according to the invention, its binding pattern can be determined analogously to the methods described for the test substances. The binding pattern determined describes the target substance phenomenologically. Are z. B. lipophilic regions present in the target substance bind corresponding binding-active substances from the characterized library, polar regions are accessible, polar substances from the library are bound. Using the determined binding pattern, the target substance can be assigned a specific point in n-dimensional space that is spanned by the individual components.
Chemischen Zielsubstanzen bzw. Testsubstanzen mit ähnlichem Bindungsverhalten werden im bibliotheksspezifischen n-dimensionalen Raum nahe beieinander liegende Punkte zugeordnet.Chemical target substances or test substances with a similar binding behavior are assigned to closely spaced points in the library-specific n-dimensional space.
Die Erfindung betrifft weiterhin ein Verfahren zur phänomenologisehen Beschreibung von Zielsubstanzen unter Verwendung einer erfindungsgemäßen charakterisierten Bibliothek aus potentiell bindungsaktiven Substanzen, bei dem die mit den zu untersuchende Zielverbindung in Wechselwirkung mit der charakterisierten Bibliothek gebracht wird und anschließend das durch die spezifischen Wechselwirkungen erzeugte Bindungsmuster der Zielsubstanz bestimmt wird. Aufgrund des ermittelten Bindungsmusters wird nach dem oben beschriebenen Verfahren der Zielsubstanz ein Punkt im n-dimensionalen Raum der charakterisierten Bibliothek zugeordnet, der das Bindungsverhalten der Zielsubstanz wiedergibt. Nun können die Testsubstanzen ermittelt werden die durch benachbarte Punkte im n-dimensionalen Raum der charakterisierten Bibliothek repräsentiert werden und aufgrund dessen ein ähnliches Bindungsverhalten zeigen. Aus den bekannten Agonisten bzw. Antagonisten der Testsubstanzen können dann physikochemische Deskriptoren identifiziert werden, die für eine spezifische Bindung mit der untersuchten Zielsubstanz förderlich sind. Ein großer Vorteil der vorliegenden Erfindung ist es, daß eine neu identifizierte Zielstruktur, für die weder Agonisten, Antagonisten oder sonstige Bindungspartner bekannt sein müsse, mit einer Gruppe von Testsubstanzen korreliert werden kann, die strukturell ähnliche Merkmale und/oder physikoehemisch ähnliche Eigenschaften besitzen. Das bedeutet umgekehrt, daß ausgehend von den strukturellen Merkmalen und physikochemischen Eigenschaften der bekannten Agonisten, Antagonisten und Nichtbindern der ähnlichen Testsubstanzen Rückschlüsse auf die physikochemischen und strukturellen Voraussetzungen potentieller Agonisten und Antagonisten der untersuchten Zielstruktur gezogen werden können. Auf der Grundlage der gewonnen Informationen kann eine gezielte Auswahl von Verbindungen vorgenommen werden die auf eine spezifische Wechselwirkung mit der zu untersuchenden Zielsubstanz untersucht werden können. Durch das erfindungsgemäße Verfahren wird folglich, im Gegensatz zum „Try and Error" - Prinzip, die zielgerichtete Auswahl von physikochemischen Deskriptoren ermöglicht, die bei der Suche nach aktiven Substanzen (Effektoren) zu einer unbekannten Zielsubstanz eingesetzt werden können, ohne daß auf ein rationales Moleküldesign zurückgegriffen werden muß.The invention further relates to a method for the phenomenological description of target substances using a characterized library of potentially binding active substances according to the invention, in which the target compound to be investigated is brought into interaction with the characterized library and subsequently the binding pattern of the target substance generated by the specific interactions is determined becomes. Based on the determined binding pattern, a point in the n-dimensional space of the characterized library is assigned to the target substance according to the method described above, which point reflects the binding behavior of the target substance. Now the test substances can be determined, which are represented by neighboring points in the n-dimensional space of the characterized library and therefore show a similar binding behavior. The known agonists or antagonists of the test substances can then be used to identify physicochemical descriptors which are conducive to specific binding with the target substance under investigation. A great advantage of the present invention is that a newly identified target structure, for which neither agonists, antagonists or other binding partners need to be known, can be correlated with a group of test substances which have structurally similar features and / or physico-chemically similar properties. Conversely, this means that, based on the structural characteristics and physicochemical properties of the known agonists, antagonists and non-binders of the similar test substances, conclusions can be drawn about the physicochemical and structural requirements of potential agonists and antagonists of the investigated target structure. On the basis of the information obtained, a targeted selection of compounds can be made which can be examined for a specific interaction with the target substance to be examined. In contrast to the “try and error” principle, the method according to the invention consequently enables the targeted selection of physicochemical descriptors that can be used in the search for active substances (effectors) to an unknown target substance, without having to rely on a rational molecular design must be used.
Interessante Zielsubstanzen sind vor allem Proteine, die dem Auftreten bestimmter Krankheiten zugeordnet werden können, dabei ist unerheblich, ob ihre dreidimensionale Struktur bekannt bzw. deren biochemisches Verhalten aufgeklärt ist.Interesting target substances are above all proteins that can be assigned to the occurrence of certain diseases. It is irrelevant whether their three-dimensional structure is known or their biochemical behavior is elucidated.
Weitere interessante Zielstrukturen sind genregulatorisch aktive DNA-Sequenzen, wie. z. B. Promotoren oder Operatoren.Other interesting target structures are gene-regulatory active DNA sequences, such as. z. B. promoters or operators.
Die nach dem oben beschriebenen Verfahren identifizierten Deskriptoren können z. B;. zur Herstellung einer kombinatorisch-synthetisch erzeugten Bank (J. M. Blaney, E. J. Martin, Current Opinion in Chemical Biology 1997, 1 , 54-59) aus potentiellen Effektoren verwendet werden. Bei der Erzeugung einer solchen Bibliothek können weitere Deskriptoren berücksichtigt werden, die bekannte chemisch-physikalische Eigenschaften besitzen, so z. B. Strukturelemente die die Membrangängigkeit von Wirkstoffen fördern, oder Strukturelemente, die die biologische oder physiologische Abbaubarkeit oder Ausscheidbarkeit verbessern (P. J. Sinko, Current Opinion in Drug Discovery & Development 1999,242-48).The descriptors identified by the method described above can e.g. B ; , to produce a combinatorial-synthetically generated bank (JM Blaney, EJ Martin, Current Opinion in Chemical Biology 1997, 1, 54-59) from potential effectors. When creating such a library, further descriptors can be taken into account, the well-known chemical-physical Have properties such. B. Structural elements that promote the membrane passage of active ingredients, or structural elements that improve the biodegradability or excretibility (PJ Sinko, Current Opinion in Drug Discovery & Development 1999, 242-48).
Ein Effektor im Sinne dieser Erfindung ist eine biologisch oder chemisch aktive Substanz, die mit der zu untersuchenden Zielsubstanz spezifisch in Wechselwirkung tritt und deren Funktion beeinflußt. Effektoren sind z. B. Inhibitoren, Aktivatoren oder Induktoren von Enzymen, Coenzymen Transkriptionsfaktoren oder Repressoren.An effector in the sense of this invention is a biologically or chemically active substance which specifically interacts with the target substance to be investigated and influences its function. Effectors are e.g. B. inhibitors, activators or inducers of enzymes, coenzymes, transcription factors or repressors.
Somit ist ein weiterer Gegenstand der Erfindung die Verwendung von Deskriptoren, die unter Einsatz einer erfindungsgemäßen charakterisierten Bibliothek ermittelt wurden, für die Herstellung einer Bibliothek aus potentiellen Effektoren, sowie die zielgerichtet erzeugten Bibliotheken zur Identifizierung von geeigneten Effektoren der zu untersuchenden Zielsubstanz.Another object of the invention is thus the use of descriptors, which were determined using a characterized library according to the invention, for the production of a library from potential effectors, and the target-generated libraries for the identification of suitable effectors of the target substance to be examined.
Geeignete Effektoren können nun mit den dem Fachmann bekannten Methoden identifiziert werden. Suitable effectors can now be identified using the methods known to those skilled in the art.

Claims

Patentansprüche: claims:
1. Verfahren zur phänomenologisehen Beschreibung von Zielmolekülen umfassend folgende Verfahrensschritte: a) kontaktieren einer zu untersuchenden Zielsubstanz mit einer charakterisierten Substanzbibliothek aus potentiell bindungsaktiven Substanzen, b) Ermittlung des Bindungsmusters der zu untersuchenden Zielsubstanz mit den bindungsaktiven Substanzen, c) Identifizierung bekannter Testsubstanzen mit einem möglichst ähnlichen Bindungsverhalten wie das der zu untersuchenden Zielsubstanz und Ermittlung der bekannten Agonisten und/oder Antagonisten dieser Testsubstanzen.1. A method for the phenomenological description of target molecules comprising the following process steps: a) contacting a target substance to be investigated with a characterized substance library of potentially binding substances, b) determining the binding pattern of the target substance to be examined with the binding-active substances, c) identifying known test substances with one if possible similar binding behavior as that of the target substance to be examined and determination of the known agonists and / or antagonists of these test substances.
2. Verfahren nach einem der vorherigen Ansprüche dadurch gekennzeichnet, daß Bibliothek mit Proteinen, Peptiden oder mit Nukleinsäuren als Testsubstanzen charakterisiert wurde.2. The method according to any one of the preceding claims, characterized in that library with proteins, peptides or with nucleic acids has been characterized as test substances.
3. Verfahren nach einem der vorherigen Ansprüche dadurch gekennzeichnet, daß die Ermittlung des Bindungsmusters massenspektrometrisch erfolgt.3. The method according to any one of the preceding claims, characterized in that the determination of the binding pattern is carried out by mass spectrometry.
4. Verfahren nach einem der vorherigen Ansprüche dadurch gekennzeichnet, daß der Vergleich des Bindungsverhaltens von Ziel- und Testsubstanz über eine Mustererkennung und einer mathematischen Auswertung nach der Clusteranalyse erfolgt.4. The method according to any one of the preceding claims, characterized in that the comparison of the binding behavior of the target and test substance via a pattern recognition and a mathematical evaluation takes place after the cluster analysis.
5. Substanzbibliothek enthaltend potentiell bindungsaktive Substanzen dadurch gekennzeichnet, daß die Bibliothek mit Testverbindungen charakterisiert ist.5. substance library containing potentially binding substances, characterized in that the library is characterized with test compounds.
6. Substanzbibliothek enthaltend potentiell bindungsaktive Substanzen dadurch gekennzeichnet, daß Substanzen enthalten sind, die bekannte therapeutische Targets binden. 6. Substance library containing potentially binding substances, characterized in that it contains substances that bind known therapeutic targets.
7. Substanzbibliothek nach Anspruch 5 oder 6 dadurch gekennzeichnet, daß die potentiell bindungsaktiven Substanzen Carbonsäuren, Amine, Ester, Aldehyde, Ketone, Acetale und Heterocyclen, wie Alkaloide, und Lipide, Saccharide, Steroide sowie andere Naturstoffe, aber auch Peptide und Proteine, wie Antikörper oder Peptoide sowie deren Homo- oder Heterodimere bzw. - multimere oder deren Agonisten und Antagonisten sind.7. substance library according to claim 5 or 6, characterized in that the potentially binding substances carboxylic acids, amines, esters, aldehydes, ketones, acetals and heterocycles, such as alkaloids, and lipids, saccharides, steroids and other natural products, but also peptides and proteins, such as Antibodies or peptoids and their homo- or heterodimers or multimers or their agonists and antagonists.
8. Substanzbibliothek nach einem der Ansprüche 5 bis 7 dadurch gekennzeichnet, daß die potentiell bindungsaktiven Substanzen vorgegebene physikochemische Eigenschaften über einen weiten Eigenschaftsbereich abdecken.8. Substance library according to one of claims 5 to 7, characterized in that the potentially binding active substances cover predetermined physicochemical properties over a wide range of properties.
9. Verwendung einer charakterisierten Substanzbibliothek gemäß den Ansprüchen 5 bis 8 zur Vorauswahl von Deskriptoren als Grundlage für den Aufbau von Bibliotheken aus potentiellen Effektoren.9. Use of a characterized substance library according to claims 5 to 8 for the preselection of descriptors as the basis for the construction of libraries from potential effectors.
10. Verwendung des Verfahrens gemäß der Ansprüche 1 bis 4 zur zielgerichteten Vorauswahl von Deskriptoren als Grundlage für den Aufbau von Bibliotheken aus potentiellen Effektoren.10. Use of the method according to claims 1 to 4 for the targeted preselection of descriptors as the basis for the construction of libraries from potential effectors.
11. Substanzbibliothek erhältlich aus Deskriptoren ermittelt nach einem Verfahren gemäß einem Ansprüche 1 bis 4 mittels kombinatorischer Synthese.11. substance library obtainable from descriptors determined by a method according to one of claims 1 to 4 by means of combinatorial synthesis.
12. Substanzbibliothek nach Anspruch 11 dadurch gekennzeichnet, daß die mittels kombinatorischer Synthese erzeugten Verbindungen durch Deskriptoren variiert sind.12. Substance library according to claim 11, characterized in that the compounds generated by combinatorial synthesis are varied by descriptors.
13. Verfahren zur Auffindung geeigneter Effektoren für eine zu untersuchende Zielsubstanz umfassend folgende Verfahrensschritte: a) generieren einer Bibliothek gemäß der Ansprüche 10 bis 12 aus potentiellen Effektoren enthaltend Strukturmerkmale von Agonisten und/oder Antagonisten ermittelt gemäß einem Verfahren nach einem der Ansprüche 1 bis 4, b) kontaktieren der zu untersuchenden Zielsubstanz mit dieser Bibliothek und c) Identifizierung von Bindungspartnern der zu untersuchenden Zielsubstanz.13. A method for finding suitable effectors for a target substance to be examined, comprising the following method steps: a) generating a library according to claims 10 to 12 from potential effectors containing structural features of agonists and / or antagonists determined according to a method according to one of claims 1 to 4, b) contacting the target substance to be examined with this library and c) identifying binding partners of the target substance to be examined.
14. Verfahren nach Anspruch 10 dadurch gekennzeichnet, daß die14. The method according to claim 10, characterized in that the
Bindungspartner anschließend auf ihre Effektoreigenschaften getestet werden. Binding partners are then tested for their effector properties.
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